With non-heme Fe oxidases and oxygenases carrying out some fundamentally very significant biological reactions, gaining an understanding of how they work is vital to being able to implement an effective treatment plan for remedy of any problem associate with one. These oxidases and oxygenases react with oxygen to form Fe-oxo intermediates which are subsequently used to carry out oxidative reactions. Due to low oxygen solubility under biological conditions, coupled with often fast off rates for O2 binding and fast decay rates of the Fe-oxo species, many of these intermediates lifetimes are quite short lived. This had made their characterization rather problematic. This proposal aims at identifying these transient Fe-oxo intermediates in a variety of non-heme Fe oxidases and oxygenases systems through utilization of enzymatically generated O2. Through utilization of the enzyme chlorite dismutase to produce oxygen on demand from its reaction with chlorite (ClO2-), higher concentrations of O2 can be achieved allowing for faster rates of intermediate formation before decay. This accumulation of intermediates then allows for their spectroscopic characterization, providing insight into how their active site configurations affect the ensuing chemistries. The specific aims of the research project are twofold: The first is to characterize Fe-oxo intermediates in systems which have been partially characterized, but due to low levels of intermediate accumulation merit further study. Such systems include the peroxo and bridged oxo intermediate in E. coli ribonucleotide reductase, and the superoxo bound intermediate in Isopenicillin N synthase and Myo-inositol oxygenase; enzymes involved in deoxyribonucleotide synthesis, penicillin production and inositol synthesis. The second specific aim is to characterize systems which are proposed to form Fe-oxo species, but due to the fleeting nature of the intermediate have yet to be detected. This includes the proposed Fe-superoxo and Fe-hydroperoxo species in (S)-2-hydroxypropylphosphonic acid epoxidase (HppE), Hydroxyethylphosphonate dioxygenase (HEPD), and Tryptophan hydroxylase (TrpH) as well as others, with the later of these enzymes playing important oxidative roles in the synthesis of the antibiotic fosfomycin (HEPD) and in the synthesis of the neurotransmitter serotonin (TrpH). Through the work of this proposal we will undoubtedly shine light onto the workings of these enzymatic systems thus giving important clues into the role of Fe-oxo species in these reactions of significant biological importance.